Heterologous protein production in a host such as a fungus is well known. EP-A-481,008 discloses production of a heterologous protein in a yeast, which is grown on glucose.
The industrial, large scale production of a heterologous protein by a host organism in a fed batch fermentation generally shows three phases.
The first phase is the batch phase which is defined as the phase wherein the cells are grown to the required concentration. In this phase the cells are grown exponentially. Models describing the batch phase assume that cells do not die, that oxygen is present in excess, and that all other conditions are such that growth can occur unlimited. This implies that in the batch phase all nutrient requirements are supplied in sufficient quantity. In summary the batch phase is the phase where cells are amplified while (heterologous) protein production is still low.
The second phase is the feed phase which is defined as the phase wherein carbon source and other requirements are fed to a fermenter in a relatively concentrated liquid stream at a precalculated rate, the “feed rate”. In this phase emphasis is on protein production by the grown cells and cell growth leading to an increase of biomass. Substrate that is fed to the fermenter is at this stage used generally for cell growth and product synthesis. The cell growth is controlled by the feed rate to obtain an optimum in cell growth and production of eterologous protein.
Eventually the third phase is reached which is defined as the decline phase, wherein limiting conditions arise. In this phase for example oxygen concentration in the fermenter is low to zero, leading in some cases to formation of ethanol. In this stage, most cells will focus on maintenance and usually product synthesis is reduced. Although cell growth may still be observed in this phase, growth is generally very limited to zero. Gradually cells may loose viability in this phase.
The production of heterologous protein on a medium comprising a common carbon source like glucose or another sugar based carbon source is satisfying until limiting conditions start to exist at the end of the feed phase. Examples of limiting conditions include reduced oxygen concentration, reduced nutrients like vitamins, carbon, nitrogen, and accumulation of toxic compounds in the growth medium.
If a fungus, especially a yeast, is grown on a medium comprising glucose as carbon source, as soon as limiting conditions arise, heterologous protein production is considerably reduced.
For yeasts grown on common medium including a sugar as carbon source the above-described phases exist in a fed-batch fermentation. Hence once the decline phase has started, specific production, which is defined as amount of heterologous protein or peptide produced per gram of biomass, is maintained or reduced. Even although cell density is high, product synthesis is hence low in the decline phase.
Another disadvantage of common media which often comprise glucose as a carbon source, is the high amount of substrate that is converted to biomass instead of conversion to product which is usually a heterologous protein in the context of this invention. Hence in such systems, a relatively high amount of biomass unavoidably accompanies the production of high levels of heterologous protein.
This high biomass leads to a viscous fermentation medium in which for example oxygen limitation easily arises.
Therefore there is a desire for a method for heterologous protein production in a host like a fungus which leads to high heterologous protein yield even under limiting conditions, where normally decline and reduced specific production would exist, whereas at the same time the specific production of the growth system is maintained or increased compared to the known growth systems.
The method of the current invention overcomes at least one of the indicated problems.